Hepatitis C virus (HCV) is a blood-borne, hepatotropic RNA virus that affects over 3% of world population with a high rate of persistent infection (50-80%) and chronic necro-inflammatory liver disease that can progress to cirrhosis and cancer. The relevance of T cells in HCV infection has been suggested by impaired HCV-specific effector T cell response in patients with chronic HCV infection and by prolonged viremia in T cell-depleted chimpanzees. The precise mechanisms for HCV-specific T cell dysfunction are not fully defined. However, there is compelling evidence in the recent literature and our preliminary data to support the relevance of immune co-stimulatory pathways (PD1, CTLA4) and immune regulatory T cells (FoxP3+ Tregs, IL10+ Tr1) in HCV-specific T cell dysfunction. In this proposal, we hypothesize that HCV- specific effector T cells are functionally suppressed by multiple mechanisms, both directly by immune costimulatory signals on effector T cells and indirectly by immune regulatory T cells. We also propose that HCV-specific T cells are further tolerized in the liver (the site of HCV infection) and that targeted inhibition of one or more immune regulatory pathways can enhance virus-specific effector function. To this end, we will examine the following 3 Aims to determine if: Aim 1. HCV-specific effector T cell dysfunction is mediated directly by immune co-inhibitory pathways and indirectly by immune regulatory T cells. Co-stimulatory receptor expression (PD- 1, CTLA4, CD28, 4-1BB) by circulating virus-specific T cells will be examined relative to their antigen-specific effector function, immune regulatory T cell frequency and phenotype (Foxp3+ or IL10+) and virological outcome in acute, chronic or resolved HCV infection, focusing the detailed analysis on study subjects that have already been recruited (with known outcomes), while enrolling new patients for sample collection. Aim 2. HCV-specific effector T cell dysfunction is accentuated in HCV-infected liver. HCV- specific T cells in the liver will be examined for antigen-specific effector function and co- stimulatory receptor expression relative to peripheral blood. We will focus on establishing the in vitro culture system of HCV-replicating hepatoma cells expressing costimulatory ligands, while collecting intrahepatic lymphocytes needed for Aim 3. Aim 3: HCV-specific effector T cell dysfunction can be reversed by modulating immune co- stimulatory pathways. The functional consequences of modulating co-stimulatory pathways or Treg-mediated suppression will be examined in peripheral HCV-specific T cells. Direct analysis using in vitro co-culture system and intrahepatic T cells will begin after the first two years, after the reagents are better established. The proposed studies will provide novel insights to underlying immunological mechanisms of T cell dysfunction and HCV persistence with potential therapeutic application.